Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase

John David Dignam,Xiaogang Qu,Jonathan B Chaires

doi:10.1074/jbc.m006840200

John David Dignam, Xiaogang Qu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m006840200

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Abstract

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.

Highlights

Aminoacyl-tRNA synthetases are a structurally diverse group of enzymes divided into two classes based on the topography of their adenylate binding sites and their modes of tRNA binding [1,2,3]
Unfolding of Glycyl-tRNA Synthetase—Examination of the denaturation of glycyl-tRNA synthetase by urea or guanidineHCl using spectroscopic techniques sensitive to protein conformation reveals a complex unfolding pathway expected of a multidomain, dimeric enzyme [48]
The results with the two proteins suggest that the N-terminal element constitutes an independent folding entity. This may be the case for E. coli seryl-tRNA synthetase [49] where the unfolding of the N-terminal coiled-coil has been examined, this element was examined while separated from the rest of the protein

Summary

EXPERIMENTAL PROCEDURES

Materials—Wild type B. mori glycyl-tRNA synthetase and a mutant lacking the first 55 N-terminal residues (N55) were expressed in E. coli BL21DE3pLysS transformed with plasmids encoding these proteins (pNADA and pNADAN55, described previously [15]). Samples were prepared for sedimentation studies by chromatography on a column of Sephadex G-50 medium equilibrated in 50 mM potassium phosphate (pH 7.2), 0.1 mM EDTA containing 0.05 mM DTT and the indicated concentrations of urea. Unfolding Experiments—Samples for spectroscopic measurements were diluted at least 100-fold into solutions containing 50 mM potassium phosphate (pH 7.2), 0.1 mM EDTA, 0.05 mM DTT and the indicated concentration of guanidine-HCl or urea. Singular value decomposition converts the data matrix into the product of a U matrix (basis spectra), an S matrix (singular values), and a V matrix (amplitude vectors) These three matrices allow for the determination of the number of spectrally significant species over a range of guanidine-HCl or urea. A simple two-state model [33] with the native protein (N) and the unfolded species (U) is described by, N7U

Sobs ϭ

SN ϩ

SNp ϩ

REACTION V

Basis spectrum

DISCUSSION